BACTERIAL GROWTH & PHYSIOLOGY II MBBS

Dr Sudheer Kher Department of Microbiology

Growth 

It is an increase in all the cell components, which ends in multiplication of cell leading to an increase in population.

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It involves - an increase in the size of the cell & an increase in the number of individual cells.

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Bacteria divide by binary fission.

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Professor Sudheer Kher

Generation time Interval of time between two cell divisions OR  The time required for a bacterium to give rise to 2 daughter cells under optimum conditions 

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Also called population doubling time.

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Professor Sudheer Kher

Generation time 

Coliform bacilli like E.coli & other medically important bacteria – 20 mins

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Tubercle bacilli – 20 hrs

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Lepra bacilli – 20 days

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Professor Sudheer Kher

Growth form in Laboratory 

Colony – formed by bacteria growing on solid media. (20-30 cell divisions) Each bacterial colony represents a clone of cells derived from a single parent cell.

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Turbidity – liquid media - 107-109 cells/ml

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Pellicle formation – thin spread over an inert surface.

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Professor Sudheer Kher

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Professor Sudheer Kher

Bacterial counts 

Growth in numbers can be studied by bacterial counts.

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2 methods – Total cell count - Viable cell count

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Professor Sudheer Kher

Total Count 

Total number of cells in the sample – living + dead.

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Can be obtained by : Direct counting under microscope using counting chambers.

1.

2.

Counting in an electronic device – Coulter counter.

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Professor Sudheer Kher

Total Count  3.

Can be obtained by : Direct counting using stained smears - by spreading a known volume of culture over a measured area of slide.

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Opacity measurements using absorptiometer/ nephalometer.

5.

Chemical assays of cell components.

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Professor Sudheer Kher

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Viable Cell Count 

Measures the number of living cells.

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Methods – Surface colony count

1. Dilution method 2. Plating method

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Number of colonies that develop after incubation gives an estimate of the viable count. Professor Sudheer Kher

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Professor Sudheer Kher

Bacterial Growth Curve 

When a bacterium is added to a suitable liquid medium & incubated, its growth follows a definite course.

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If bacteria counts are made at intervals after inoculation & plotted in relation to time, a growth curve is obtained.

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Shows 4 phases : Lag, Log or Exponential, Stationary & phase of Decline.

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Professor Sudheer Kher

Phases of Growth Curve 

Lag phase – No increase in number but there may be an increase in the size of the cell.

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Log OR Exponential phase – cells start dividing and their number increases exponentially.

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Professor Sudheer Kher

Phases of Growth Curve 

Stationary phase – cell division stops due to depletion of nutrients & accumulation of toxic products. - the number of progeny cells is just enough to replace the dying cells - equilibrium exists between dying cells and the newly formed cells, so viable count remains stationary

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Phase of Decline – population decreases due to the death of cells – autolytic enzymes.

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Professor Sudheer Kher

4/15/2012

Professor Sudheer Kher

Morphological & Physiological alterations during growth 

Lag phase – maximum cell size towards the end of lag phase.

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Log phase – smaller cells, stain uniformly

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Stationary phase – irregular staining, sporulation and production of exotoxins & antibiotics

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Phase of Decline –involution forms(with ageing) 4/15/2012

Professor Sudheer Kher

Factors Affecting Bacterial Growth Temperature Atmosphere – O2 & CO2 H-ion concentration Moisture & drying Osmotic effects Radiation Mechanical & sonic stress. 4/15/2012

Professor Sudheer Kher

Temperature 

Vary in their temperature requirements.

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Temperature range – growth does not occur above the maximum or below the minimum.

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Optimum Temperature – growth occurs best, 37ºC for most pathogenic bacteria.

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Professor Sudheer Kher

Temperature 

Mesophilic – grows best between 25ºC and 40ºC. e.g. most bacterial pathogens

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Psychrophilic (cold loving) – grows best below 20ºC e.g. Flavobacterium spps

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Thermophilic – grows best at high temp, 5558ºC e.g. Bacillus stearothermophilus

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Professor Sudheer Kher

Atmosphere  1.

Depending on the O2 requirement, bacteria are divided into : Strict (Obligate) Aerobes – require O2 for growth e.g. Pseudomonas aeruginosa

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Strict (Obligate) Anaerobes – grow in the absence of O2 & may even die on exposure to O2 e.g. Bacteroides fragilis

3.

Microaerophilic – grow best in the presence of low oxygen levels e.g. Campylobacter spp, Helicobacter spp

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Professor Sudheer Kher

Atmosphere 4.

Facultative anaerobe – aerobic but can also grow in the absence of O2 e.g. Staphylococcus spps

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Aerotolerant anaerobe – anaerobic, but tolerates exposure to O2 e.g. Clostridium perfringens

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Capnophilic organism – requires high CO2 levels eg Neisseria spps

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Professor Sudheer Kher

H-ion Concentration 

pH range, optimum pH

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Neutral or slightly alkaline pH (7.2 – 7.6) – majority of pathogenic bacteria grow best.

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Lactobacilli – acidic pH

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Vibrio cholerae – alkaline pH

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Professor Sudheer Kher

Moisture & Drying 

Water – essential ingredient of bacterial protoplasm. Hence drying is lethal to cells.

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Effect of drying varies : T.pallidum – highly sensitive Staphylococci sp– stand for months

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Spores – resistant to dessication, may survive for several decades.

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Professor Sudheer Kher

Osmotic effects 

More tolerant to osmotic variation due to mechanical strength of their cell walls.

Radiation 

X rays & gamma rays exposure – lethal

Mechanical & Sonic Stress 

May be ruptured by mechanical stress.

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Professor Sudheer Kher

Bacterial Nutrition 

Water constitutes 80% of the total weight of bacterial cells.

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Proteins, polysaccharides, lipids, nucleic acids, mucopeptides & low molecular weight compounds make up the remaining 20%.

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For growth & multiplication, the minimum nutritional requirements are water, a source of carbon, a source of nitrogen & some inorganic salts.

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Professor Sudheer Kher

Classification of Bacteria Based on Nutritional Requirement 

Phototrophs – Bacteria which derive their energy from sunlight.

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Chemotrophs – Bacteria which derive energy from chemical reactions. Organotrophs : require organic sources of hydrogen 2. Lithotrophs : require inorganic sources of hydrogen like NH3, H2S 1.

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Professor Sudheer Kher

Classification of Bacteria Based on Nutritional Requirement 

Based on the utilization of carbon compounds, bacteria are classified as : 1.

Autotrophs – can synthesise all their organic compounds, able to utilise atmospheric CO2 & N2. No medical importance.

2.

Heterotrophs – unable to synthesise their own metabolites & depend on preformed organic compounds.

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Professor Sudheer Kher

Growth Factors 

Some bacteria require certain organic compounds in minute quantities – Growth Factors OR Bacterial Vitamins.

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It can be : Essential – when growth does not occur in their absence. 2. Accessory – when they enhance growth, without being absolutely necessary for it. 1.

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Professor Sudheer Kher

Growth Factors  

Identical with mammalian nutrition Vitamin B complex – 1. 2. 3. 4. 5. 6.

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thiamine riboflavine nicotinic acid pyridoxine folic acid & Vit.B 12

Professor Sudheer Kher

Summary  

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Growth is defined as an increase in number. Bacterial growth in liquid culture has four separate stages Lag phase is the time before cell growth begins In exponential phase, cell begin to divide Cells stop increasing in number during stationary phase Environmental factors greatly effect the growth of microbes

4/15/2012

Professor Sudheer Kher